Scalable, Accessible eDNA

Can we remove reliance on cold chains from the metaprobe 2.0 eDNA framework?

Project Overview

Environmental DNA (eDNA) is genetic material that is shed from macro-organisms. Recent years have seen the piloting of eDNA barcoding in British marine waters, and the validation of the metaprobe 2.0 in the passive filtration and collection of eDNA.

A key selling feature of the metaprobe eDNA sampling device is it's ease of deployment, but current protocols still require the inclusion of a cold chain. A cold chain is the necessity of having your samples on ice or in a —20 (or greater) freezer until the stage of extraction. This is a key limitation on the application of the metaprobe in the wild, and so over the past year I've worked with a Masters student, Carla Leone, here at Exeter to test the efficacy of removing the cold chain from the metaprobe work flow.

The meta-what now?

The metaprobe!

Let's refresh: a metaprobe is a 3D printed sphere, secured with cable ties. And within the sphere are 3 rolled up pieces of cotton-gauze. Each gauze is a sampling replicate, meaning there are 3 sampling replicates are within 1 device

Metaprobes break the mould because, unlike common eDNA sampling methods for the marine environment, metaprobes capitalise on the natural flow of seawater and don't require active filtration via peristaltic pump.

Once brought back on board, the gauze rolls, are removed from the metaprobe and stablisied in 98.9% ethanol.

Then, in the lab, we subsample the gauze, and extract the DNA from it's tissue using a modified protocol and readily available DNA extraction kits.

A bit more background

As part of my PhD we advanced the research exploring the capacity of metaprobes. We showed they work effectively relative to standard dip-sampling and filtration over medical grade filters via vacuum pressure.

This was the workflow for that study, and what I'd like you to notice here is the inclusion of the cold chain - the bit highlighted in pink. This inclusion of a cold chain seems harmless but is actually logistically challenging and in some locations, impossible.

We invision the metaprobe as a tool for industry, academics, monitoring agencies, and citizen scientists alike, in the Global North and SSouthuth. And so we wanted to test whether this cold chain was absolutely necessary.

Study design

We designed an experiment varying the storage temperature and duration of metaprobes.

Carla collected a 35L sample of water from the sea, and exposed all samples to the same water for 60 minutes. Samples were then separated into their experimental groups, in a factorial design, designated to one of two temperatures groups, Ambient and –20°C, and one of five duration groups. Duration was recorded as the time between the sampling day and the date of extraction. The first was the conducted the same day as sampling. the last 8 weeks after sampling.

Then, to provide some information on the performance and preservation of DNA over time, we looked at two methods: double stranded DNA concentration quantification and metabarcoding. We looked at the concentration of double stranded DNA as a total measure of retention and release. And species richness and community composition as indicated by the metabarcoding analyses as a measure of performance.

The Exeter Sequencing Facility performed the metabarcoding, pooling together both the MiFish U primer targeting teleosts and the MiFish E primer targeting elasmobranchs.

Results?

Coming soon!

Thank you to Carla!

Thank you to Carla Leone, the MSc student on this project. We're pursuing publicaiton of Carla's thesis so you'll have to wait for the results for now.

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